The overall goal is to provide a structural basis for understanding the mechanisms of initiation and elongation by DNA and RNA polymerases and the mechanisms by which these polymerases assure that the correct nucleotide is inserted with high accuracy. This objective will be achieved by determining the crystal structures of polymerases complexed with functionally associated proteins and bound to appropriate DNA or RNA substrates. We aim to establish the structure of a replication fork complex that includes the DNA polymerase, sliding clamp, primase and possibly helicase bound to an appropriate DNA fork substrate using the proteins from either T4 or RB69 phage. The structures of smaller binary or ternary complexes will also be examined in solution by X-ray scattering, crystallized and probed by genetic experiments. Additionally, the structure of the DNA B helicase complexed with the DNA G primase from Thermus aquaticus bound to DNA will be pursued. The structural mechanisms by which DNA lesion bypass polymerases are able to bypass DNA lesions will be pursued by co-crystallizing the DinB lesion bypass DNA polymerase from Sulfolobus sofactaricus with a DNA substrate containing a lesion in the template strand. A third family of DNA polymerase to be examined is phi29 DNA p0I, which initiates DNA synthesis with a protein subunit. A fifth family of polymerases to be studied is T7 RNA polymerase, a homologue of the pot I family of DNA polymerases. To understand the transition from the initiation phase of RNA synthesis at a promoter to the elongation phase, the structure of the T7 RNA polymerase complexed with 30 base-pairs of DNA, 17 nucleotide RNA transcript and an incoming ribonucleoside triphosphate analogue will be determined. A sixth family of polymerases to be studied includes the CCA-adding enzyme, an RNA potymerase that adds CCA to the 3' ends of tRNA in an untemplated fashion. To establish how the accurate addition of CCA is accomplished at one active site without the use of a template, the co-crystal structure of this enzyme with tRNA acceptor stem analogues missing the 3' terminal A, CA or CCA complexed with a nonreactive analogue of the next incoming ribonucleoside triphosphate will be determined.